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Biophysical Journal Volume 97 September 2009 1709–1718 1709

Structural Changes to Monomeric CuZn Superoxide Dismutase Causedby the Familial Amyotrophic Lateral Sclerosis-Associated Mutation A4V

Tom Schmidlin,† Brian K. Kennedy,† and Valerie Daggett†‡*†Department of Biochemistry and ‡Department of Bioengineering, University of Washington, Seattle, Washington

ABSTRACT Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron degenerative disease, and the inherited form,familial ALS (fALS), has been linked to over 100 different point mutations scattered throughout the Cu-Zn superoxide dismutaseprotein (SOD1). The disease is likely due to a toxic gain of function caused by the misfolding, oligomerization, and eventualaggregation of mutant SOD1, but it is not yet understood how the structurally diverse mutations result in a common diseasephenotype. The behavior of the apo-monomer fALS-associated mutant protein A4V was explored using molecular-dynamicssimulations to elucidate characteristic structural changes to the protein that may allow the mutant form to improperly associatewith other monomer subunits. Simulations showed that the mutant protein is less stable than the WT protein overall, with shifts inresidue-residue contacts that lead to destabilization of the dimer and metal-binding sites, and stabilization of nonnative contactsthat leads to a misfolded state. These findings provide a unifying explanation for disparate experimental observations, allowa better understanding of alterations of residue contacts that accompany loss of SOD1 structural integrity, and suggest siteswhere compensatory changes may stabilize the mutant structure.

INTRODUCTION

A subset of familial amyotrophic lateral sclerosis (fALS)

cases has been tied to mutations in the Cu-Zn superoxide dis-

mutase protein (SOD1). Though the mechanism of toxicity

has been under intense scrutiny for 15 years, it remains

largely undetermined. One current model involves the mis-

folding of the mutant protein (1), which results in a new

structural species that directly confers toxicity and/or seques-

ters cellular machinery that is important for protein homeo-

stasis, such as chaperones or proteasome components.

SOD1 is normally responsible for the disproportionation

of superoxide to molecular oxygen and hydrogen peroxide,

in which one superoxide molecule is oxidized and then

another is reduced by copper in the active site of the enzyme.

Superoxide is a naturally occurring byproduct of respiration.

Although SOD1 is abundant and ubiquitous in the cyto-

plasm, it has also been found in the mitochondrial intermem-

brane space. The concentrations are especially high in certain

subcellular locations, such as motor neuron axons (2).

Only minimal changes have been observed in the crystal

structure of fully metallated disease-associated mutant forms

of SOD1 in comparison to wild-type (WT) SOD1. Many

SOD1 mutants display lower melting temperatures than their

WT counterparts, regardless of the metallation state (3), and

they unfold more easily with urea or guanidine-HCL (4).

However, some of the metal-binding mutants have higher

melting temperatures than WT (5). Both sporadic and

familial forms of ALS show evidence of cytoplasmic inclu-

sion bodies—deposits that are characteristic of many neuro-

degenerative disorders, including Huntington’s disease,

Submitted December 12, 2008, and accepted for publication June 15, 2009.

*Correspondence: [email protected]

Editor: Kathleen B. Hall.

� 2009 by the Biophysical Society

0006-3495/09/09/1709/10 $2.00

Alzheimer’s disease, and Parkinson’s disease (6)—and

aggregates have been found in mouse models of the disease

(7–9). The familial ALS aggregates contain SOD1, neurofi-

lament proteins, ubiquitin, and a host of other cellular

components, but it is not clear whether zinc or copper is

present (10). The current thinking is that the aggregation

is a cellular protective mechanism and the most toxic form

is either a misfolded monomer or a soluble oligomeric

species or protofibril (6). Similar models have been proposed

for other neurodegenerative diseases (11).

The unfolding and aggregation pathway of the protein

involves the dissociation of the dimer and loss of metal

binding, followed by the subsequent oligomeric assembly

of the protein (12–14). It is also possible that the aggregates

are favored when the amount of misfolded protein reaches

a point where the ubiquitin proteolytic machinery becomes

unable to handle the load (15,16). Although the aforemen-

tioned experimental approaches have been enlightening,

there are other opportunities to investigate the effects of

mutation on the protein. An atomic-level look at the protein

dynamics through molecular-dynamics (MD) simulations

can be informative in determining the effect the mutations

have on the structure of the protein, allowing investigation

of how changing one residue to another can create a ripple

effect through the protein that eventually affects dimeriza-

tion, metal binding, and/or overall protein stability.

SOD1 conforms to the Greek key b-barrel folding

topology, and each monomer subunit of the homodimer

binds one copper and one zinc ion. Although the crystal

structures of several ALS-causing mutants have been solved,

the structure of WT SOD1 in solution differs from the crystal

structure (17). These average structures are very informative;

however, proteins are dynamic and important conforma-

tional states may have low sampling rates.

doi: 10.1016/j.bpj.2009.06.043

mailto:[email protected]

1710 Schmidlin et al.

Previous MD studies have included short (<1 ns) simula-

tions of WT bovine and human SOD1 dimers (18–20), as

well as comparisons of simulations of the WT monomer

and dimer (21,22). In addition, 5 ns simulations of WT and

mutant (A4V, G37R, and H46R) dimers and monomers

(23) have provided the first direct comparison of mutant

and WT MD simulations. These results indicate that altered

long-range communication within the protein structure could

be an underlying cause of aggregation of mutant SOD1 in

fALS. Also, a recent study examined several 100 ns simula-

tions of WT apo and holo monomers and dimers, and the

effects of disulfide bonds on SOD1 stability (24).

Here we report findings from multiple long (60 ns) simu-

lations of WT and A4V apo (demetallated) monomers to

explore the dominant patterns of structural changes upon

mutation in the monomer after loss of metallation. A4V is

very close to the dimer interface and represents one of the

most common and most lethal mutations, with rapid disease

progression and death occurring on average at 1.4 years vs.

3–5 years for other mutants (25). These simulations reveal

significant changes in the mutant protein structure, which

is discussed in terms of potential effects on dimer destabili-

zation, loss of metallation, and the aberrant oligomerization

of the misfolded monomer.

MATERIALS AND METHODS

Models

The starting structure for WT SOD1 monomer was obtained from a 1.8 A

crystal structure of human SOD1 (1HL5 Chain A) (26). The A4V SOD1

starting structure was obtained from a 1.9 A crystal structure of the A4V

mutant of human SOD1 (1UXM Chain A) (17). Before energy minimization

was performed, the Cu2þ and Zn2þ ions were removed and H63 was left un-

protonated. The ionization states of the amino acids corresponded to neutral

pH (Asp�, Glu�, His0, Lysþ, and Argþ). The C57-C146 disulfide bond was

intact to match in vitro studies.

The holo 1HL5 structure was chosen over the apo 1HL4 structure for

a variety of reasons. In brief, of the four SOD1 proteins available in the

1HL4 tetramer, chains C and D are missing many residues (26 and 28 out

of 153, respectively). Chains A and B are missing many heavy atoms

(18 and 12 atoms, respectively) and they also have bound zinc. The model-

building required to render these structures suitable for simulation would

be quite extensive, and the resultant starting structure would be inferior to

the 1HL5 structure used.

MD simulations

Simulations of apo WT and A4V SOD1 were performed with the in lucem

Molecular Mechanics (ilmm) (27) simulation software using protocols

described elsewhere (27–32). In brief, the simulations included all hydro-

gens and explicit flexible three-center waters (29). The protein was solvated

in a box extending at least 10 A from any protein atom, with the solvent

density set to the experimental value at 310 K of 0.993 g/mL (33). Periodic

boundary conditions were employed to minimize edge effects. The microca-

nonical constant number of molecules, volume, and energy (NVE) ensemble

was employed. A 10 A force-shifted cutoff range was used for all nonbonded

atom interactions, and the interaction pair list was updated every three steps.

The potential energy functions were used to propagate MD trajectories with

a 2 fs time step, and structures were saved every 1 ps for analysis. Three

Biophysical Journal 97(6) 1709–1718

60 ns simulations were run for each WT and A4V apoprotein at 310 K,

yielding ~180,000 structures each for analysis. The long simulation time

allows the different simulations to reach a ‘‘converged’’ state for each mono-

mer in solution with enough sampling of the state to be statistically sound.

Analyses

Except where noted, all analyses were performed over all structures. The

Define Secondary Structure of Proteins (DSSP) algorithm (34) was used

to assign secondary structure based on hydrogen-bond energies. The

DSSP data used to generate the graphs were sampled at 1 ps intervals;

however, conformational time-based populations from DSSP were calcu-

lated at 100 ps intervals.

The CONGENEAL structural dissimilarity scores (35) for each simula-

tion were calculated at each time point based on the minimized structure

of the protein. CONGENEAL is based on the weighted distance maps of

two structures, such that the weight is higher for atoms that are closer

together. Each element of the matrix is calculated using the equation

wi,j ¼ di,j�p, where w is the weight, d is the Ca distance between the two

residues, and p ¼ 2.

Differences between the total contact time of residue pairs in the WT and

A4V simulations were considered to be statistically significant if the differ-

ence in the averages of the two different types of simulations was greater

than the combined standard deviation (SD) of those values. Atoms were

considered to be in contact if the heavy atom distance was <4.6 A, except

for C-C, where the cutoff was 5.4 A. Protein images were produced using

UCSF Chimera (36).

RESULTS AND DISCUSSION

Simulations of the fALS-associated SOD1 mutant A4V

showed the effects of the mutation on the kinetic stability

of the protein when compared with WT under similar condi-

tions (neutral pH, 310 K, apo, monomer). For each WT and

mutant protein, three simulations were run for 60.5–63.0 ns

and then analyzed. The differences between the two

sequences may be illustrated in a number of ways to provide

clues for the previously reported effects of this mutation on

dimer stability (14) and metal binding (37,38).

Overall protein instability

The simulations reveal that during the course of simulation,

the A4V mutant protein undergoes larger structural changes

than WT. An examination of the Ca root mean-square devi-

ation (RMSD) (see Fig. S1, A and B, in the Supporting

Material), total solvent accessible surface area (SASA)

(Fig. S1, C and D), radius of gyration (Fig. S1, E and F),

and CONGENEAL structural dissimilarity score (Fig. S1,

G and H) for the simulations shows a general increase in

all of these measures across the simulations for the mutant

versus WT. The smaller changes in the SASA and radius

of gyration data indicate that the mutant protein sample

conformations are somewhat different but still fairly

compact. A breakdown of the various components that

comprise the total SASA of the protein reveals that each of

the components of the total SASA is generally higher in

the mutant than in the WT (data not shown). Overall, larger

changes in the structure are observed for A4V than for WT.

fALS Mutation A4V Alters SOD1 Structure 1711

Perturbations of residues normally at the dimerinterface

Dimerization of the individual monomer subunits increases

the stability of the SOD1 enzyme (39), and thus mutations

that destabilize the dimer may decrease the overall protein

stability. Dimer destabilization may be viewed in two

ways: instability in the dimer itself, or a decreased affinity

for dimerization by the individual monomer subunits. Pertur-

bations of the native dimer interface residues may either

prevent the initial dimerization or decrease the lifetime of

the dimer. Although the effects of the mutation on the dimer

would be better studied using dimer simulations, our intent

was to study the A4V-associated structural changes in the

monomer that occur after the known loss of dimerization,

which may have implications for the ability of the dimer to

reform.

One way to measure perturbations of the protein in MD

simulations is to examine the changes in contacts between

residues over time. A minimum increase or decrease in total

contact time of 50% over the entire simulation between any

residue pair in the protein was used as a cutoff, allowing for

the creation of a map showing the major changes in intramo-

nomer residue-residue contacts as the effects propagated

from one point of interest to another (Fig. 1 A). There is an

overall increase in the total contact time of the dimer inter-

face residues (50–53, 114, and 148–153) (17) with other

residues in the A4V simulations in comparison with WT,

although there is a significant decrease (51%) in total contact

time between residues I149 and F20 (Fig. 1, B and C). These

intramonomer contacts serve to bring the N- and C-termini of

the protein into tighter association in the mutant simulations,

potentially impairing the ability of the A4V mutant to main-

tain dimerization.

Residues S105 and C111 form an isolated b-bridge struc-

ture at the beginning of all simulations (Fig. 2, A and B). A

bridge is essentially defined as an elementary hydrogen-

bonding pattern that is a building block for a- or b-sheet.

In the mutant runs, this b-bridge is unstable and each simu-

lation adopts a fairly stable p-helix comprised of residues

L106–H110 after some period of time (Fig. 2 B). The time

it takes to form this helix varies from one simulation to the

next, and it is variably stable after it forms. In contrast, no

helix is found in this region during any of the WT simula-

tions, and the b-bridge is retained to varying degrees in the

three runs (Fig. 2 A). Although they are not directly involved

in the dimer interface, these residues have an impact on it

through the contacts formed or lost during the course of

the simulations. For instance, there is a large loss of contacts

between L106 and K3, and an increase in contacts between

K3 and dimer interface residue G150, as described above.

Additionally, there is a loss of contacts between nearby

S105 and I112, I112 with F20, and F20 with dimer interface

residue I149 (Fig. 1, B and C). These structural changes near

the dimer interface provide a reasonable mechanism to

FIGURE 1 Contact differences between WT and A4V simulations. (A)

All changes in residue-residue contact time > 50% of the total simulation

time, relative to the WT simulations. (B) Changes in contact time between

various residues mapped onto the minimized crystal structure of the mutant

protein. (C) Changes in contact time between various residues displayed for

clarity.



FIGURE 2 Protein secondary structure. Hydrogen-bond

analysis and secondary structure assignment of representa-

tive (A) WT and (B) A4V simulations using DSSP. (C) The

starting structure of the WT protein with the b-strands

labeled.

explain the substantial destabilization of the A4V mutant

SOD1 dimer (17), which is the first step of the proposed olig-

omerization pathway.

Perturbations of the metal-binding sites

Copper-binding site

The residues involved in coordinating the metal (Fig. S2) are

distal to the A4V mutation, yet they show greater movement

during simulation in the mutant than in WT. The Ca root

mean-square fluctuation (RMSF) (Fig. 3, A and B) and Ca

RMSD (not shown) plots show higher values for these resi-

dues, although the distances between the Cu2þ ion-binding

atoms are not significantly different between the WT and

mutant proteins (Fig. 3 C). This is not surprising given the

known role of the Cu2þ ion in the enzymatic reaction, and

the normal level of catalytic activity of this mutant (37,40).

Motion of the electrostatic loop in WT apo dimer simula-

tions was previously reported (22) and we also observe this

motion in our monomer simulations. However, the helix in

the electrostatic loop near the copper-binding site is more

stable and has less apparent motion in the WT form

(Fig. S3 A) than in the mutant (Fig. S3B). The rotation of

this helix may be explained by a large decrease in the con-

tacts between the residues of the helix and the body of the

protein, specifically residue H63, which coordinates both

Zn2þ and Cu2þ binding. Table S1 shows that H63 has

greatly reduced total contact times with S134 (57%

FIGURE 3 Changes to metal-binding residues. (A)

Ca-RMSF by residue for three each WT (light gray, shown

in red online) and A4V (dark gray, blue online) simulation.

(B) Differences in Ca-RMSF are mapped onto the protein

structure. Regions with a higher RMSF in the mutant than

in the WT are shown in dark gray (blue online), and regions

with higher RMSF in the WT than in the mutant are shown

in light gray (red online). White indicates no difference.

The four residues displayed are the Zn2þ-binding residues.

H71, H80, and D83 each show higher RMSF and RMSD

(not shown) in the A4V mutant simulations than in the

WT. (C) Average pairwise distances between the Cu2þ-

binding atoms for three simulations of WT (diamonds)and A4V (squares) proteins. In each case the error bars

represent the SD of the three simulations, and in each

case the error bars overlap. The WT crystal structure

distances are shown as triangles. Mutant crystal structure

distances differ from WT by <0.2 A in all cases, including

those shown in panel D (not shown). (D) Average pairwise

distances between the Zn2þ-binding atoms for three simu-

lations of WT (diamonds) and A4V (squares) proteins. In

each case the error bars represent the SD of the three simu-

lations. There are significant differences between the

distances of the pairs: 63ND1-71ND1, 63ND1-83OD1,

71ND1-80ND1, and 80ND1-83OD1. Triangles are as in

panel C.



reduction), T135 (43%), T137 (27%), and G138 (58%), and

H120 has less contact with G138 (57%) and N139 (75%)

during the mutant simulations compared to WT. Residues

in this region (residues 122–140) that differ by>50% in total

contact time between the WT and A4V simulations are

shown in Fig. 4. Toward the end of the third WT simulation,

the helix moves into a similar position as seen in the mutant,

which may mean that this motion is not an important effect

of the mutation. However, the apo form of the protein is

less stable (3,5,39,41), and this may simply be a consequence

of the lower stability. The mutant protein spends far more

time with the helix in the new position than does WT. There

is also greater movement of the C-terminal residues in WT

than in the mutant, as discussed above.

The native a-helix in the electrostatic loop, comprised of

residues E132–T137 (Fig. 5 A), is somewhat more stable in

the WT simulations than in the mutant runs. Toward the end

of one WT run at ~56 ns, the N-terminus of the helix

extends to include neighboring residues through K128. In

another WT run, the helix converts to p-helix intermittently

from ~7 to 23.5 ns, spending ~57% of this time in a p-helical

structure before returning more stably to the a-helix struc-

ture. The mutant simulations are somewhat different. In

one case, the a-helix in this region is very stable. In another,

a fairly stable p-helix forms that lasts from ~27.5 ns until the

FIGURE 4 Contact differences of the helix near the Cu2þ-binding site.

All changes are relative to WT simulations. (A) Changes in contact time

between various residues mapped onto the minimized crystal structure of

the mutant protein. (B) Changes in contact time between various residues

displayed for clarity.

end of the run (60 ns). In the third simulation, the helix

falls apart after only 3.5 ns. It returns stably for over 20 ns

beginning around 27 ns into the run, but after that it comes

apart again and does not refold before the end of the simu-

lation.

Although the A4V mutant has roughly WT levels of dis-

mutase activity per copper, it also has only 50% as much

copper bound (37). The destabilization of the electrostatic

loop observed by MD may affect the ability of the copper

chaperone to adequately load the copper ion into the binding

site. Alternatively, the deformations of the loop may affect

the protein’s ability to hold onto the metal, accounting for

the drop in observed copper binding.

FIGURE 5 Helix near the Zn2þ-binding residues. Locations of the native

(E132-T137) and nonnative (G73-E77) helix are shown in WT (A) in the

starting structure and (B) at 11.6 ns. Three of the zinc-binding residues are

shown as spheres.



Zinc-binding site

The change in average distance between some of the zinc-

binding atoms is significant in the mutant simulations

(Fig. 3 D). Although there is no difference in the distance

between atoms 63ND1 and 80ND1 or between 71ND1 and

83OD1, there is a difference in the distances between

63ND1 and both 71ND1 and 83OD1, as well as between

80ND1 and both 71ND1 and 83OD1. These results indicate

that residues H63 and H80 move together in the mutant, as

do H71 and D83. This motion and the increased atom-

atom distance may prevent the mutant from properly binding

Zn2þ, consistent with the experimentally observed 30-fold

decrease in zinc affinity in the A4V mutant (38). Zinc

binding is known to stabilize the protein (3,5,41), and loss

of metal binding precedes oligomerization. Recent evidence

suggests that loss of zinc binding occurs simultaneously with

dimer dissociation on the millisecond timescale of the exper-

imental work (42).

A nonnative helix is formed around residues 73–77 in WT

(Fig. 5 B), a stretch that is unstructured in the crystal struc-

ture. Although these residues are not themselves involved

with zinc binding, they are close to the zinc-binding residues

H71 and H80. The different WT runs show a helical structure

forming in this region for varying amounts of time. The

nature of the helix also varies, alternating between a-helix,

p-helix, and 3/10 helix; however, the formation of some

helical structure at this location is consistent across all three

simulations, whereas no significant amount of helix is found

in this location during any of the mutant simulations. Struc-

tural stability in this region may be increased by the helix in

the WT simulations, accounting for the smaller changes in

the zinc-coordinating atom distances compared to the mutant

simulations. Movements of the zinc-binding loop were noted

in a previous work (22), but no mention was made of a helix

in this area.

Contact network analysis revealed a potential propagation

pathway for structural changes across the protein, which may

also lead to the observed differences in the pairwise distances

of the Zn2þ-binding residues (Fig. 6 A). A maximum of

seven contacts are needed for the four Zn2þ residues in the

map (H63, H71, H80, and D83) at a 45% cutoff. The three

Cu2þ-binding residues (H48, H63, and H120) required

a maximum of nine contacts, whereas the fourth residue

(H46) could not be included at that cutoff.

In Fig. 6 B this contact network is mapped onto the struc-

ture to illustrate how the A4V mutation causes a ripple effect

through the protein. The contacts shift between residues, and

the changes propagate throughout the protein. For example,

G27 loses contact with S105 and moves to S102. The loss of

contact between G27 and S105 causes S105 to lose contact

with residues I112 and P28, both of which then lose contact

with residue I104. Loss of contact between residues I112 and

I104 causes residue I112 to come into contact with residue

P66, a contact that almost never occurs in the WT simula-


tions (2% 5 3.46%). Residue I104 then loses contact with

G82 and has a significant increase in contact with residues

H80 and R79, and R79 loses contact with H71. Note that

H80 and H71 are zinc-binding residues. A list of the contacts

gained and lost during the simulations, as reflected in Fig. 6,

is provided in Table S1.

Through this type of analysis, we are able to identify key

residues between the mutation site and the site of interest,

such as residue I104, which serves as a branch point on

the map leading to all three of the Zn2þ-binding residues.

This residue could be mutated to halt the propagation of

the changes and, it is hoped, eliminate the phenotypes caused

by the A4V mutation. However, even though I104 is highly

conserved (43), there is a known fALS mutation at this

residue: I104F. If I104 serves as a key residue in maintaining

FIGURE 6 Contact differences between WT and A4V simulations. All

changes shown represent a minimum of a 45% absolute change in contact

time relative to WT simulations. (A) Map of the changes in contact time dis-

played for clarity. (B) The starting structure of the A4V mutant shown with

selected changes in contact between residues. The changes displayed repre-

sent the shortest pathways of change between the mutation site and the

metal-binding residues. Metal-binding residue not shown (Cu2þ: H46) did

not meet the threshold of 45% change in absolute contact time between

WT and A4V simulations.


SOD1 stability, mutation at this site may only further exac-

erbate the instability of the monomer.

Potential effects on oligomeric assembly

Exposure of cysteines

Previous research indicates that C6 and C111 play a role in

SOD1 aggregation, although reports vary. It is not clear

whether both Cys residues are important for oligomerization

(44,45), C111 is important but C6 is not (46), or C6

and C111 play some role but are not the only important

factors (47).

Our results indicate that C111 has a ~20% higher average

solvent exposure in A4V compared to WT (69.1 A2 for

A4V vs. 57.1 A2 for WT), and the close association of b8

with b1 in the mutant simulations effectively prevents a signif-

icant change in solvent accessibility for C6 (1.1 A2 for A4V

vs. 1.5 A2 for WT). The increase in solvent accessibility for

C111 was predicted via Carr-Purcell-Meiboom-Gill 15N

nuclear spin relaxation dispersion measurements by K. Tei-

lum, M. H. Smith, E. Schulz, G. Solomentsev, M. Oliveberg,

and M. Akke (unpublished). This solvent exposure of the free

cysteine C111 may allow for nonnative monomer-monomer

interactions.

b-Sheet instability

The starting structures of both the WT (Fig. 2 C) and mutant

proteins contain b-strand comprising residues G85–A89

(strand b5) and V94–D101 (strand b6). b5 is an edge strand

of the sheet formed with strands b4, b7, and b8, whereas

strand b6 is an edge strand of the sheet formed with

strands b1, b2, and b3. Strands b5 and b6 are more stable

in WT than in mutant simulations (Fig. 2, A and B). Previous

MD simulations also found destabilization of b5 and b6 (23).

In the first two WT runs, the b-sheets are largely retained

at b5 and b6, with some loss of strand from the N-terminal

end of strand b6. In the third simulation, strand b6 loses

most hydrogen bonds with b3 by 26 ns, although some

b-bridge is retained through the end of the simulation. In

the A4V simulations, it is b5 that is less stable. In the first

mutant run, the hydrogen bonds formed with b4 have largely

disappeared after only 2.2 ns, although some b-bridges

appear infrequently. Strand b6 is much more stable, retaining

some b-sheet contacts for ~97% of the simulation and at a

minimum some b-bridges for >99% of the 60 ns simulation.

The second simulation reveals a loss of b-sheet beginning

with b5 (residues G85–A89), which loses most of the

b-structure after only a few nanoseconds. Some degree of

b-structure is retained in this region for nearly 40 ns,

however, alternating between fragments of b-sheet and

b-bridges during that time. After 40 ns, what little structure

is found in this area is a in nature, mostly a-bridges formed

between residues N86 on b5 and H43 on b4. Strand b6 also

begins to lose structure early in the simulation; however, the

ends of the strand remain in contact with b3 for much of the

first 40 ns of simulation. By 52.5 ns, there is a complete loss

of b-structure in b6, but by 44.6 ns residue D96 begins to

form frequent a-bridge contact with S34 on neighboring

b3. This contact is present ~94% of the time through the

end of the simulation. The third mutant simulation also

rapidly loses b-sheet contacts involving b5, as well as

a slower but substantial loss of b-sheet contacts from b6.

Strand b5 loses most of its b-structure by 4 ns, whereas b6

loses b-structure at ~30 ns. In each of the mutant simulations,

the loss of b-sheet contacts in b5 and b6 are accompanied by

some loss of contacts in neighboring strands b4 and b3,

respectively; however, each simulation retains b-sheet in

these regions through the end of the runs. The observed

destabilization of b5 and b6 is consistent with previous

MD studies (23).

The typical forms of edge b-strands protect proteins from

edge-edge aggregation (48). The perturbation and loss of the

native edge strands in the mutant simulations provide

another potential mechanism by which the mutant monomers

can improperly interact with other monomers. The b-barrel,

specifically b3 and b4 (49) and b5 and b6, was previously

identified experimentally as a potential hotspot for local un-

folding leading to aggregation in fALS cases (50), and our

results are in good agreement with recent findings of b-strand

destabilization of apo SOD1 monomers (24).

a-Strand and a-bridges

A hydrogen-bond analysis of the first A4V run shows non-

helical local a-structure beginning after 31 ns and continuing

at some level through the end of the simulation. This local

a-structure, when alternating between aL and aR, can give

rise to strands similar to b-strands, but in this case the main

chain carbonyl oxygens are aligned on one side and the amide

hydrogens along the other, rather than alternating. We refer

to this structure as a-sheet. From 31.1 ns to 51.3 ns, a-strand

or bridges are present in the area of residues F50–G51 and

S59–A60, near the dimer interface, for ~44% of the time. At

this point there is a ~6 ns period during which there is

no sampling of this structure; sampling then resumes at

~57.6 ns and continues for roughly 64% of the time through

the end of the run. A representative structure (32.8 ns) was

chosen for further analysis of the a-sheet hydrogen-bond

energies, particularly between residues 49–52 and 58–61

(Fig. 7). Note that residues G51 and D52 are dimer interface

residues.

In the second mutant simulation, intermittent a-bridges

form between residues G51 and A60, H43 and N86, and

S34 and D96, beginning at different time points but

continuing through the end of the simulation. The a-bridge

between residues G51 and A60 forms first, initially appear-

ing at ~6.6 ns into the simulation and persisting for ~44%

of the time. The a-bridge between residues H43 and N86

forms next at ~40 ns of the run and is present ~40% of the

time through the end of the run. Finally, the a-bridge

between residues S34 and D96 does not form until ~45 ns



FIGURE 7 a-Sheet in the mutant simulations. A repre-

sentative structure of the A4V simulations shows one

location of a-sheet formation. In the right panel, the back-

ground structure and atoms have been removed for clarity.

into the run, but it is present ~94% of the time thereafter.

None of these a-bridges require either of the others to be

present for formation. In the third A4V run, a-sheet again

forms between residues F50–G51 and S59–A60, much like

in the first mutant run. There is an a-bridge between residues

G51 and A60 beginning around 4 ns of the simulation and

continuing nearly 73% of the time through 8.4 ns of the

simulation. At this point, it breaks and no significant amount

of a-bridge or a-sheet appears until after nearly 27 ns of

simulation time. After this time, there is an a-bridge or

a-sheet present in this region >83% of the time through

the end of the run.

In contrast to A4V, the a-bridge is barely populated

(<5%) in the second WT simulation, and none is found in

either of the other two WT runs. When we examine the

data by (4, j) angles, we can see that there is no a-strand

comprised of four or more consecutive residues in any of

the three neutral pH WT simulations that were run (data

not shown); however, it is present in all three of the A4V

simulations. In the first A4V simulation, this strand is

predominantly found from residues T88 to D92, forming

in the loop between b5 and b6. The other two simulations

form a-strand in different locations, specifically residues

L42–F45 in the second simulation (in strand b4) and at

a lower level in the third simulation residues E40–H43

(beginning in the loop between b3 and b4 and continuing

into b4). Some a-strand is also formed at residues V103–

L106 in the second simulation, which is part of the b-barrel

crossover loop between strands b6 and b7. This change may

be permitted due to the significant changes in the contacts of

residues V103–S105 in all of the mutant simulations, as dis-

cussed above.

It is not clear what role, if any, the presence of a-bridge or

a-sheet may play in protein aggregation, although it has been

observed in simulations of amyloidogenic proteins (32,

51–53), leading to the suggestion that aggregation can be

mediated by a-sheet (54). This structure is frequently seen

in the A4V mutant SOD1 simulations and conspicuously

absent from the WT simulations. As such, a-sheet could

provide a catalyst for aberrant oligomerization.


CONCLUSIONS

MD simulations of the fALS-associated SOD1 mutant A4V

in comparison with WT simulations indicate significant

structural differences. We found lower overall stability in

the mutant by a variety of measures, as well as perturbations

of important dimer interface residues that explain the

reduced dimerization observed experimentally. The zinc-

binding site was disrupted, which could account for the

reported 30-fold decrease in zinc-binding affinity in the

mutant. This may be caused by a failure of the mutant to

form a stabilizing a-helix in the zinc-binding loop. The helix

in the electrostatic loop also undergoes greater conforma-

tional changes in the mutant simulations. It may be that the

movement of this helix and the lack of helix in the zinc-

binding loop contribute to the loss of b-strand observed in

the mutant simulations. We also observed increased solvent

exposure of a free cysteine residue and the presence of

a-sheet in the A4V simulations, but not in the WT simula-

tions. Taken together, these results provide possible explana-

tions for dimer destabilization, loss of metallation, and

oligomerization of the mutant protein.

SUPPORTING MATERIAL

One table and three figures are available at http://www.biophysj.org/

biophysj/supplemental/S0006-3495(09)1219-3.

This study was supported by a Genetic Approaches to Aging Training Grant

from the Nathan Shock Center of Excellence in the Basic Biology of Aging

(AG 00057 to T.S.) and a grant from the National Institutes of Health

(GM 81407 to V.D.).

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http://www.biophysj.org/biophysj/supplemental/S0006-3495(09)1219-3


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